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Sheng-Cai Lin

Publications and source records attributed to Sheng-Cai Lin.

At least 19 recordsLinked to original sources
PubMed2007

Daxx cooperates with the Axin/HIPK2/p53 complex to induce cell death.

Daxx, a death domain-associated protein, has been implicated in proapoptosis, antiapoptosis, and transcriptional regulation. Many factors known to play critically important roles in controlling apoptosis and gene transcription have been shown to associate with Daxx, including the Ser/Thr protein kinase HIPK2, promyelocytic leukemia protein, histone deacetylases, and the chromatin remodeling protein ATRX. Although it is clear that Daxx may exert multiple functions, the underlying mechanisms remain far from clear. Here, we show that Axin, originally identified for its scaffolding role to control beta-catenin levels in Wnt signaling, strongly associates with Daxx at endogenous levels. The Daxx/Axin complex formation is enhanced by UV irradiation. Axin tethers Daxx to the tumor suppressor p53, and cooperates with Daxx, but not DaxxDeltaAxin, which is unable to interact with Axin, to stimulate HIPK2-mediated Ser(46) phosphorylation and transcriptional activity of p53. Interestingly, Axin and Daxx seem to selectively activate p53 target genes, with strong activation of PUMA, but not p21 or Bax. Daxx-stimulated p53 transcriptional activity was significantly diminished by small interfering RNA against Axin; Daxx fails to inhibit colony formation in Axin(-/-) cells. Moreover, UV-induced cell death was attenuated by the knockdown of Axin and Daxx. All these results show that Daxx cooperates with Axin to stimulate p53, and implicate a direct role for Axin, HIPK2, and p53 in the proapoptotic function of Daxx. We have hence unraveled a novel aspect of p53 activation and shed new light on the ultimate understanding of the Daxx protein, perhaps most pertinently, in relation to stress-induced cell death.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Differential requirement of MKK4 and MKK7 in JNK activation by distinct scaffold proteins.

Different scaffold proteins play distinct roles in various signaling pathways by recruiting different downstream molecules. Here, using MKK4(-/-) and MKK4(-/-)/7(-/-) murine embryonic fibroblast cells, we examined differential employment of MKK4 and MKK7 by scaffold proteins Axin, Dvl, and Epstein-Barr virus latent membrane protein-1 (LMP-1) in mediating JNK activation. We present evidence that Axin depends mainly on MKK7 for activation of JNK, while Dvl depends almost equally on MKK4 and MKK7 for JNK activation, In contrast, LMP-1-induced JNK activation is primarily dependent on MKK4. Our results demonstrate that Axin, Dvl, and LMP-1 differentially utilize MKK4 and MKK7 for JNK activation.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Axin is a scaffold protein in TGF-beta signaling that promotes degradation of Smad7 by Arkadia.

TGF-beta signaling involves a wide array of signaling molecules and multiple controlling events. Scaffold proteins create a functional proximity of signaling molecules and control the specificity of signal transduction. While many components involved in the TGF-beta pathway have been elucidated, little is known about how those components are coordinated by scaffold proteins. Here, we show that Axin activates TGF-beta signaling by forming a multimeric complex consisting of Smad7 and ubiquitin E3 ligase Arkadia. Axin depends on Arkadia to facilitate TGF-beta signaling, as their small interfering RNAs reciprocally abolished the stimulatory effect on TGF-beta signaling. Specific knockdown of Axin or Arkadia revealed that Axin and Arkadia cooperate with each other in promoting Smad7 ubiquitination. Pulse-chase experiments further illustrated that Axin significantly decreased the half-life of Smad7. Axin also induces nuclear export of Smad7. Interestingly, Axin associates with Arkadia and Smad7 independently of TGF-beta signal, in contrast to its transient association with inactive Smad3. However, coexpression of Wnt-1 reduced Smad7 ubiquitination by downregulating Axin levels, underscoring the importance of Axin as an intrinsic regulator in TGF-beta signaling.

Active Transport, Cell Nucleus↗
PubMed2006

Tob1 controls dorsal development of zebrafish embryos by antagonizing maternal beta-catenin transcriptional activity.

Maternal beta-catenin and Nodal signals are essential for the formation of the dorsal organizer, which, in turn, induces neural and other dorsal tissue development in vertebrate embryos. Tob (Transducer of ErbB2) proteins possess antiproliferative properties and are known to influence BMP signaling, but their relationship to other signaling pathways and to embryonic patterning in general was unclear. In this study, we demonstrate that zebrafish tob1a is required for correct dorsoventral patterning. Mechanistically, Tob1a inhibits beta-catenin transcriptional activity by physically associating with beta-catenin and preventing the formation of beta-catenin/LEF1 complexes. Although Tob1a can also inhibit the transcriptional activity of the Nodal effector Smad3, its role in limiting dorsal development is executed primarily by antagonizing the beta-catenin signal. We further demonstrate that Tob family members across species share similar biochemical properties and biological activities.

Animals↗
PubMed2006

[Effect of exogenous RGS16 gene transfection on growth of glioma cell line C6].

BACKGROUND & OBJECTIVE: Wild type p53 could induce RGS16 mRNA expression, and RGS16 protein is related with carcinogenesis of glioma. This study was designed to investigate the effects of exogenous RGS16 gene transfection on the growth of rat glioma cell line C6. METHODS: A eukaryotic expression plasmid pIRES2-EGFP-RGS16 was constructed, and transfected into C6 cells. The cells were selected by G418. The expression of enhanced green fluorescent protein (EGFP) was observed under fluorescent microscope, and the expression of RGS16 mRNA and protein was detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. The effects of RGS16 gene on cell cycle progression, cell growth, and proliferation of C6 cells were measured by flow cytometry, cell growth curve, and clonogenic assay in plate. RESULTS: C6-RGS16 and C6-GFP cells, which separately expressed RGS16 and pIRES2-EGFP vector, were constructed successfully. S phase proportion was significantly higher in C6-RGS16 cells than in C6-GFP and C6 cells (28.5% vs. 18.9% and 14.3%, P<0.05); C6-RGS16 cells grew faster than C6-GFP and C6 cells. The clone formation rate was significantly lower in C6-RGS16 cells than in C6-GFP and C6 cells (12% vs. 25% and 25%, P<0.05). CONCLUSION: Exogenous RGS16 gene could promote cell cycle progression of glioma C6 cells from G1 phase to S phase, and accelerate cell growth, but can't increase the clone formation.

Animals↗
PubMed2005

Involvement of microRNA in AU-rich element-mediated mRNA instability.

AU-rich elements (AREs) in the 3' untranslated region (UTR) of unstable mRNAs dictate their degradation. An RNAi-based screen performed in Drosophila S2 cells has revealed that Dicer1, Argonaute1 (Ago1) and Ago2, components involved in microRNA (miRNA) processing and function, are required for the rapid decay of mRNA containing AREs of tumor necrosis factor-alpha. The requirement for Dicer in the instability of ARE-containing mRNA (ARE-RNA) was confirmed in HeLa cells. We further observed that miR16, a human miRNA containing an UAAAUAUU sequence that is complementary to the ARE sequence, is required for ARE-RNA turnover. The role of miR16 in ARE-RNA decay is sequence-specific and requires the ARE binding protein tristetraprolin (TTP). TTP does not directly bind to miR16 but interacts through association with Ago/eiF2C family members to complex with miR16 and assists in the targeting of ARE. miRNA targeting of ARE, therefore, appears to be an essential step in ARE-mediated mRNA degradation.

3' Untranslated Regions↗
PubMed2005

Distinct roles of basal steady-state and induced H-ferritin in tumor necrosis factor-induced death in L929 cells.

Tumor necrosis factor (TNF) alpha is a cytokine capable of inducing caspase-dependent (apoptotic) cell death in some cells and caspase-independent (necrosis-like) cell death in others. Here, using a mutagenesis screen for genes critical in TNF-induced death in L929 cells, we have found that H-ferritin deficiency is responsible for TNF resistance in a mutant line and that, upon treatment with TNF, this line fails to elevate levels of labile iron pool (LIP), critical for TNF-induced reactive oxygen species (ROS) production and ROS-dependent cell death. Since we found that TNF-induced LIP in L929 cells is primarily furnished by intracellular storage iron, the lesser induction of LIP in H-ferritin-deficient cells results from a reduction of intracellular iron storage caused by less H-ferritin. Different from some other cell lines, the H-ferritin gene in L929 cells is not TNF inducible; however, when H-ferritin is expressed in L929 cells under a TNF-inducible system, the TNF-induced LIP and subsequent ROS production and cell death were all prevented. Thus, LIP is a common denominator of ferritin both in the enhancement of cell death by basal steady-state H-ferritin and in protection against cell death by induced H-ferritin, thereby acting as a key determinant of TNF-induced cell death.

Animals↗
PubMed2004

Axin contains three separable domains that confer intramolecular, homodimeric, and heterodimeric interactions involved in distinct functions.

Axin is a major scaffold protein, interacting with diverse molecules involved in a number of signaling pathways. Axin can undergo dimer/oligomerization via its DIX domain. Here we show that whereas deletion of the DIX domain at the C terminus rendered Axin incapable of forming dimer, a larger deletion of the C-terminal region restored the ability of Axin to form dimers. Detailed analyses revealed that Axin actually contains two separate domains (D and I) in addition to the DIX domain for homodimerization. The D, I, and DIX domains alone can form homodimers. Interestingly, D and I domains strongly interact with each other, suggesting that Axin can form an intramolecular structure through D and I interaction in the absence of DIX. We also found that DIX-DIX homodimeric interaction is weak but that point mutations in the DIX domain abolished Axin homodimerization. We propose a model to suggest that Axin forms homodimeric interactions through three domains, D, I, and DIX. More importantly, lack of DIX-DIX interaction caused by point mutations in the DIX domain or deletion causes Axin to form an intramolecular loop through the D and I domains, disallowing homodimer formation. Ccd1 interacts with Axin D domain yet fails to interact with AxinDeltaDIX, confirming that D is masked after D-I looping. The Axin mutants that are defective in homodimer formation fail to activate JNK but have no effect on beta-catenin signaling. Our findings have thus provided a structural basis of conformational changes in Axin, which may underlie the diversity of Axin functions.

Axin Protein↗
PubMed2004

Axin stimulates p53 functions by activation of HIPK2 kinase through multimeric complex formation.

Axin and p53 are tumor suppressors, controlling cell growth, apoptosis, and development. We show that Axin interacts with homeodomain-interacting protein kinase-2 (HIPK2), which is linked to UV-induced p53-dependent apoptosis by interacting with, and phosphorylating Ser 46 of, p53. In addition to association with p53 via HIPK2, Axin contains a separate domain that directly interacts with p53 at their physiological concentrations. Axin stimulates p53-dependent reporter transcription in 293 cells, but not in 293T, H1299, or SaOS-2 cells that are defective in p53 signaling. Axin, but not AxindeltaHIPK2, activates HIPK2-mediated p53 phosphorylation at Ser 46, facilitating p53-dependent transcriptional activity and apoptosis. Specific knockdown of Axin by siRNA reduced UV-induced Ser-46 phosphorylation and apoptosis. Kinase-dead HIPK2 reduced Axin-induced p53-dependent transcriptional activity, indicating that Axin stimulates p53 function through HIPK2 kinase activity. Interestingly, HIPK2deltaAxin that lacks its Axin-binding region acts as a dominant-positive form in p53 activation, suggesting that the Axin-binding region of HIPK2 is a putative autoinhibitory domain. These results show that Axin acts as a tumor suppressor by facilitating p53 function through integration of multiple factors.

Apoptosis↗
PubMed2004

RXRalpha acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells.

Retinoid X receptor (RXR) plays a crucial role in the cross talk between retinoid receptors and other hormone receptors including the orphan receptor TR3, forming different heterodimers that transduce diverse steroid/thyroid hormone signaling. Here we show that RXRalpha exhibits nucleocytoplasmic shuttling in MGC80-3 gastric cancer cells and that RXRalpha shuttling is energy-dependent through a nuclear pore complex (NPC)-mediated pathway for its import and an intact DNA binding domain-mediated pathway for its export. In the presence of its ligand 9-cis retinoic acid, RXRalpha was almost exclusively located in the cytoplasm. More importantly, we also show that RXRalpha acts as a carrier to assist translocation of TR3, which plays an important role in apoptosis. Both RXRalpha and TR3 colocalized in the nucleus; however, upon stimulation by 9-cis retinoic acid they cotranslocated to the cytoplasm and then localized in the mitochondria. TR3 export depends on RXRalpha, as in living cells GFP-TR3 alone did not result in export from the nucleus even in the presence of 9-cis retinoic acid, whereas GFP-TR3 cotransfected with RXRalpha was exported out of the nucleus in response to 9-cis retinoic acid. Moreover, specific reduction of RXRalpha levels caused by anti-sense RXRalpha abolished TR3 nuclear export. In contrast, specific knockdown of TR3 by antisense-TR3 or TR3-siRNA did not affect RXRalpha shuttling. These results indicate that RXRalpha is responsible for TR3 nucleocytoplasmic translocation, which is facilitated by the RXRalpha ligand 9-cis retinoic acid. In addition, mitochondrial TR3, but not RXRalpha, was critical for apoptosis, as TR3 mutants that were distributed in the mitochondria induced apoptosis in the presence or absence of 9-cis retinoic acid. These data reveal a novel aspect of RXRalpha function, in which it acts as a carrier for nucleocytoplasmic translocation of orphan receptors.

3T3 Cells↗
PubMed2004

Beta-actin is required for mitochondria clustering and ROS generation in TNF-induced, caspase-independent cell death.

Tumor necrosis factor (TNF)-alpha induces caspase-independent cell death in the fibrosarcoma cell line L929. This cell death has a necrotic phenotype and is dependent on production of reactive oxygen species (ROS) in the mitochondria. To identify genes involved in this TNF-induced, ROS-dependent cell death pathway, we utilized retrovirus insertion-mediated random mutagenesis to generate TNF-resistant L929 cell lines and we subsequently identified genes whose mutations are responsible for the TNF-resistant phenotype. In one such resistant line, beta-actin was disrupted by viral insertion, and subsequent reconstitution of beta-actin expression levels in the mutant line Actin(mut) restored its sensitivity to TNF. Resistance to TNF in Actin(mut) cells is signal specific since the sensitivity to other death stimuli is either unchanged or even increased. Comparable NF-kappaB activation and p38 phosphorylation in TNF-treated wild-type and Actin(mut) cells also indicates that reduced expression of actin only selectively blocked some of the TNF-induced cellular changes. Actin cleavage involved in apoptosis does not occur in TNF-treated L929 cell death, as in HeLa cells. Consistent over-expression of a caspase-cleaved product, a 15 kDa actin fragment, had no effect on TNF-induced necrosis of L929 cell. By contrast, TNF-induced mitochondria clustering and ROS production were dramatically reduced in Actin(mut) cells, indicating that actin-deficiency-mediated TNF resistance is most likely due to impaired mitochondrial responses to TNF stimulation. Our findings suggest that a full complement of actin is required for transduction of a cell death signal to mitochondria in TNF-treated L929 cells.

Actins↗
PubMed2004

The DIX domain protein coiled-coil-DIX1 inhibits c-Jun N-terminal kinase activation by Axin and dishevelled through distinct mechanisms.

Axin, Ccd1 (coiled-coil-DIX1), and dishevelled (Dvl or Dsh) are three known DIX domain proteins that play important roles in Wnt signaling. In addition, Dvl and Axin can activate the mitogen-activated protein kinase JNK via distinct mechanisms, through interaction with MEKK1/4 and Rac GTPase, respectively. Axin utilizes two distinct domains for interaction with MEKK1 and MEKK4. JNK activation by Axin is regulated by several factors in the Wnt pathway, whereas little is known about cross-regulation of Dvl-mediated JNK activation. In the present study, we have investigated whether Ccd1 could play a regulatory role in Axin- and Dvl-mediated JNK activation. Here we show that Ccd1 drastically inhibited JNK activation both by Axin and by Dvl. Although DIX domains are sufficient for dimer formation between Dvl and Ccd1, Ccd1 also required its coiled-coil domain for inhibition of JNK activation by Dvl. Interestingly, Rac remained associated with Dvl heterodimerized with Ccd1. How Ccd1 blocks Rac/Dvl signaling to JNK is unclear. In contrast, Axin, when complexed with Ccd1, did not bind to MEKK1. Furthermore, Ccd1 physically interacted with MEKK4 in their physiological concentrations and prevented MEKK4 from binding to Axin. Reduction of Ccd1 protein by small interfering RNA could elevate JNK signaling as assayed with an AP1-dependent transcriptional reporter. We have therefore demonstrated that Ccd1 inhibits Axin-mediated JNK activation by simultaneously adopting two distinct mechanisms, one through conformational changes that disallow MEKK1 binding and the other via direct sequestration of MEKK4.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Detection of point mutations of the Axin1 gene in colorectal cancers.

Axin is a recently identified tumor suppressor that plays an important role in liver and colon cancers. To gain further insights into the structure and function of Axin in controlling cell growth, we analyzed 54 colorectal cancer tissues for mutations in AXIN1 gene. We employed PCR amplification with 23 sets of primers against introns that encompassed the whole coding region of AXIN1 followed by single-strand conformation polymorphism (SSCP) analysis. After subcloning and sequencing analysis of the reamplified DNA from the aberrant bands, we found, in addition to 3 silent mutations, 6 missense point mutations in different functionally important regions. The missense mutation rate is hence 11%, suggesting that Axin deficiency may contribute to the onset of colorectal tumorigenesis.

Adenocarcinoma↗
PubMed2003

Cidea-deficient mice have lean phenotype and are resistant to obesity.

The thermogenic activity of brown adipose tissue (BAT), important for adaptive thermogenesis and energy expenditure, is mediated by the mitochondrial uncoupling protein1 (Ucp1) that uncouples ATP generation and dissipates the energy as heat. We show here that Cidea, a protein of unknown function sharing sequence similarity with the N-terminal region of DNA fragmentation factors Dffb and Dffa, is expressed at high levels in BAT. Cidea-null mice had higher metabolic rate, lipolysis in BAT and core body temperature when subjected to cold treatment. Notably, Cidea-null mice are lean and resistant to diet-induced obesity and diabetes. Furthermore, we provide evidence that the role of Cidea in regulating thermogenesis, lipolysis and obesity may be mediated in part through its direct suppression of Ucp1 activity. Our data thus indicate a role for Cidea in regulating energy balance and adiposity.

Adipose Tissue, Brown↗
PubMed2003

Axin utilizes distinct regions for competitive MEKK1 and MEKK4 binding and JNK activation.

Axin is a multidomain protein that plays a critical role in Wnt signaling, serving as a scaffold for down-regulation of beta-catenin. It also activates the JNK mitogen-activated protein kinase by binding to MEKK1. However, it is intriguing that Axin requires several additional elements for JNK activation, including a requirement for homodimerization, sumoylation at the extreme C-terminal sites, and a region in the protein phosphatase 2A-binding domain. In our present study, we have shown that another MEKK family member, MEKK4, also binds to Axin in vivo and mediates Axin-induced JNK activation. Surprisingly MEKK4 binds to a region distinct from the MEKK1-binding site. Dominant negative mutant of MEKK4 attenuates the JNK activation by Axin. Activation of JNK by Axin in MEKK1-/- mouse embryonic fibroblast cells supports the idea that another MEKK can mediate Axin-induced JNK activation. Expression of specific small interfering RNA against MEKK4 effectively attenuates JNK activation by the MEKK1 binding-defective Axin mutant in 293T cells and inhibits JNK activation by wild-type Axin in MEKK1-/- cells, confirming that MEKK4 is indeed another mitogen-activated protein kinase kinase kinase that is specifically involved in Axin-mediated JNK activation independently of MEKK1. We have also identified an additional domain between MEKK1- and MEKK4-binding sites as being required for JNK activation by Axin. MEKK1 and MEKK4 compete for Axin binding even though they bind to sites far apart, suggesting that Axin may selectively bind to MEKK1 or MEKK4 depending on distinct signals or cellular context. Our findings will provide new insights into how scaffold proteins mediate ultimate activation of different mitogen-activated protein kinase kinase kinases.

Axin Protein↗
PubMed2003

Ephrin-B1 reverse signaling activates JNK through a novel mechanism that is independent of tyrosine phosphorylation.

Eph receptors and their cognate ligand ephrins play important roles in various biological processes such as cell migration, axon guidance, and synaptic plasticity. One characteristic feature of the Eph-ephrin signal transduction is that, upon interaction with the receptor, the transmembrane B-class ephrins become tyrosine-phosphorylated and transduce intracellular signals that lead to reorganization of the cytoskeleton. Although in vitro and genetic studies have demonstrated unequivocally the significance of this reverse signaling, the underlying mechanism remains unclear. We report here that transfection of ephrin-B1 into 293 cells resulted in robust increase in JNK activity, whereas expression of truncated ephrin-B1 lacking the cytoplasmic domain had a negligible effect, indicating that the induction of JNK activity was attributed mainly to the reverse signaling. The ephrin-B1-mediated JNK activation was reduced significantly by dominant-negative TAK1, MKK4, or MKK7. Ephrin-B1 over-expressing 293 cells became rounded in morphology. Surprisingly, ephrin-B1 that lacked all six intracellular tyrosine residues still triggered JNK activation and rounding morphology of the transfected cells. Consistent with these observations, activation of JNK and the resulting morphological changes mediated by ephrin-B1 could be abolished by the JNK inhibitor SP600125 but not the Src inhibitor PP2. Taken together, our findings have identified a novel reverse signaling pathway transduced by ephrin-B1, which is independent of tyrosine phosphorylation but involves the activation of JNK through TAK1 and MKK4/MKK7 and leads to changes in cell morphology.

Animals↗
PubMed2002

SUMO-1 modification of the C-terminal KVEKVD of Axin is required for JNK activation but has no effect on Wnt signaling.

Axin is a multifunctional protein, regulating Wnt signaling and the c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK) pathway as well as tumorigenesis. In the present study, we found that Axin interacts with three SUMO-1 (small ubiquitin-related modifier) conjugating enzymes 3 (E3), PIAS1, PIASxbeta, and PIASy. The extreme C-terminal six amino acid residues of Axin are critical for the Axin/E3 interaction as deletion of the six residues (AxinDeltaC6) completely abolished the ability of Axin to interact with E3 enzymes. AxinDeltaC6 also failed to activate JNK, although it was intact in both its interaction with MEKK1 and homodimerization. Consistent with the presence of a doublet of the KV(E/D) sumoylation consensus motif at the C-terminal end (KVEKVD), we found that Axin is heavily sumoylated. Deletion of the C-terminal six amino acids drastically reduced sumoylation, indicating that the C-terminal six amino acids stretch is the main sumoylation site for Axin. Sumoylation-defective mutants failed to activate JNK but effectively destabilized beta-catenin and attenuated LEF1 transcriptional activity. In addition, we show that dominant negative Axin mutants blocked PIAS-mediated JNK activation, in accordance with the requirement of sumoylation for Axin-mediated JNK activation. Taken together, we demonstrate that sumoylation plays a role for Axin to function in the JNK pathway.

Amino Acid Sequence↗